CHEMISTRY AND BIOLOGY OF NON-HEME IRON OXYGENASES

非血红素铁加氧酶的化学和生物学

• 批准号: 6386590
• 负责人: ELLEN L NEIDLE
• 金额: $ 18.53万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-01 至 2003-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6386590
• 关键词: Mossbauer spectrometry; Neisseriaceae; X ray crystallography; bacterial proteins; bioengineering /biomedical engineering; catalyst; circular magnetic dichroism; electron spin resonance spectroscopy; enzyme activity; enzyme structure; enzyme substrate; iron sulfur protein; nucleic acid sequence; o aminobenzoate; oxygenases; phenotype; protein binding; protein sequence; stoichiometry

DESCRIPTION: (adapted from applicant's abstract) This project proposes a comprehensive approach to understanding the chemistry and biology of aromatic ring-hydroxylating dioxygenases from the soil bacteria, Acinetobacter sp. ADP1. These enzymes catalyze the (cis) dihydroxylation of aromatic rings and, in so doing, incorporate both atoms of O2 into the ring. The proposal addresses fundamental questions for this class of enzymes including catalytic mechanism(s), the roles of the metal centers, and the molecular determinants of substrate preferences, and will investigate a previously uncharacterized aromatic ring-hydroxylating enzyme, anthranilate 1,2-dioxygenase (Ant) comparing catalytic and structural properties to those of the related benzoate 1,2-dioxygenase (Ben) from the same organism. Both of these are 2-component systems consisting of a A3B3 hexameric oxygenase containing a Rieske iron center and a mononuclear ferrous center in the A subunit, and a reductase protein containing flavin and Fe/S centers. The mononuclear center carries out the oxygenase reaction while the Rieske center acts as a redox carrier. In order to address the question of substrate specificity, the principal investigator proposes to use an approach consisting of random mutation and in vivo selection to isolate Ben enzymes that have been modified for the utilization of anthranilate as an efficient substrate. Comparison of amino acid sequences of several mutated dioxygenases resulting from this procedure with those of the wild-type enzymes, in conjunction with structural and spectroscopic data will allow an understanding of the features responsible for substrate specificity. A catalytic mechanism for these enzymes and several tests of the mechanism are also proposed.

描述：（改编自申请人的摘要）该项目提出了 理解芳香族化学和生物学的综合方法 来自土壤细菌不动杆菌的环羟基化双加氧酶。 ADP1。 这些酶催化芳环的（顺式）二羟基化，因此 这样做，将 O2 的两个原子都纳入环中。该提案涉及 这类酶的基本问题包括催化 机制、金属中心的作用以及分子决定因素 底物偏好，并将研究以前未表征的 芳香环羟基化酶、邻氨基苯甲酸1,2-双加氧酶（Ant） 与相关苯甲酸酯的催化和结构特性比较 来自同一生物体的 1,2-双加氧酶 (Ben)。这两个都是 2 组件 由含有 Rieske 铁的 A3B3 六聚加氧酶组成的系统 A 亚基中的中心和单核亚铁中心，以及还原酶 含有黄素和Fe/S中心的蛋白质。单核中心进行 加氧酶反应，而里斯克中心充当氧化还原载体。在 为了解决底物特异性的问题，主要 研究人员建议使用一种由随机突变和 体内选择以分离经过修饰的 Ben 酶 利用邻氨基苯甲酸盐作为有效底物。氨基酸比较 由该过程产生的几种突变双加氧酶的序列 那些野生型酶，连同结构和 光谱数据将有助于了解负责的特征 底物特异性。这些酶和几种酶的催化机制 还建议对该机制进行测试。